Clinical Cancer Research 2016, Nov;22(21):5349-5361

Biased Expression of the FOXP3Δ3 Isoform in Aggressive Bladder Cancer Mediates Differentiation and Cisplatin Chemotherapy Resistance  

Hanwei Zhang1,2, Kris Prado1, Kelvin Zhang3, Elizabeth M. Peek4, Jane Lee1, Xiaoyan Wang5, Jiaoti Huang6, Gang Li5,7, Matteo Pellegrini8, and Arnold I. Chin1,2,4,7

1Department of Urology, 2Broad Stem Cell Research Center, 3Department of Biological Chemistry, 4Molecular Biology Institute, 5Department of Biostatistics, 6Department of Pathology, 7Jonsson Comprehensive Cancer Center, and 8Molecular, Cell, and Developmental Biology, UCLA, Los Angeles, CA 90095, USA

 

Abstract:

Purpose: The transcriptional regulation mediating cancer cell differentiation into distinct molecular subtypes and modulating sensitivity to existing treatments is an enticing therapeutic target. Our objective was to characterize the ability of the forkhead/winged transcription factor Foxp3 to modulate the differentiation of bladder cancer.

Experimental Design: Expression of Foxp3 was analyzed by immunohistochemistry in a tumor microarray of 587 samples and overall survival in a subset of 187 patients following radical cystectomy. Functional assays were performed in SW780 and HT1376 cell lines in vitro and in vivo, and gene expression profiling performed by RNA-Seq. Validation was undertaken using gene expression profiles of 131 patients from The Cancer Genome Atlas (TCGA) consortium in bladder cancer.

Results: FOXP3 expression correlates with bladder cancer stage and inversely with overall survival, with biased expression of the FOXP3Δ3 isoform. Functional assays of FOXP3Δ3 demonstrated resistance to chemotherapy in vitro, while subcutaneous xenografts overexpressing FOXP3Δ3 developed larger and more poorly differentiated bladder cancers. RNA expression profiling revealed a unique FOXP3Δ3 gene signature supporting a role in chemotherapy resistance. Accordingly, knockdown of Foxp3 by small interfering RNA in HT1376 cells conferred sensitivity to cisplatin- and gemcitabine-induced cytotoxicity. Validation in TCGA dataset demonstrated increased expression of FOXP3 in subtypes II-IV and skewing of molecular subtypes based on FOXP3Δ3-specific gene expression.

Conclusions: (i) Biased expression of the FOXP3Δ3 isoform in bladder cancer inversely correlates with overall survival, (ii) FOXP3Δ3 induces a unique gene program that mediates cancer differentiation, and (iii) FOXP3Δ3 may augment chemotherapy resistance.

  

Supplement:

Bladder cancer is the 5th most commonly diagnosed cancer in the United States with a mortality of over 16,000 per year. About 25% of patients with newly diagnosed bladder cancer have muscle-invasive disease, which often requires systemic therapy and aggressive local-regional control. First-line systemic therapy utilizes cisplatin-based chemotherapy, which has been the mainstay for decades, with the use of recently FDA-approved anti-PD-1/PD-L1 checkpoint inhibitors in cisplatin-ineligible or second-line settings.

 

Cisplatin-based chemotherapy has a 40 to 60% response rate in bladder cancer patients, however durable responses are the minority at approximately 5%1. Chemotherapy resistance is a major challenge with no predictive biomarkers available. Recent genomic characterization of muscle-invasive bladder cancer has led to the development of molecular subtypes, with certain bladder subtypes more resistant or sensitive to chemotherapy2,3. We hypothesized that inducing cancer cell differentiation may be a strategy to overcome chemotherapy resistance. Our recent manuscript investigates the role of the forkhead/winged helix transcription factor FOXP3 in this context.

 

FOXP3 is composed of 11 coding exons with a structure consisting of a N-terminus repressor domain, zinc finger, leucine zipper, and a C-terminus forkhead domain. While best known as a key transcription factor driving T regulatory cells, FOXP3 expression has been shown to be important in multiple epithelial lineages and have previously been ascribed tumor suppressor gene functions in animal models through genetic deletions4,5.

 

In contrast, we have shown that an isoform of FOXP3, lacking exon 3 encoding a repressor domain, is progressively enriched in higher stages of bladder cancer and inversely correlated to survival following radical cystectomy. We showed that FOXP3Δ3 imparts unique gene transcription in the absence of a repressor domain and is enriched in CD44+CD49f+ cells signifying the cancer initiating cell population. We observed induction of multiple drug resistance genes and discovered that FOXP3Δ3 is both induced by cisplatin as well as mediates cisplatin-based chemotherapy resistance. Furthermore, forced expression of FOXP3Δ3 appears to skew bladder cancer differentiation to distinct subtypes.

 

Our findings suggested to us a mechanism whereby augmented FOXP3Δ3 expression in aggressive bladder cancer can induce a transcriptional program that can mediate bladder cancer differentiation that promotes chemotherapy resistance in the cancer initiating population. Treatment with cisplatin and other chemotherapies may further augment FOXP3Δ3 expression and drive treatment resistance (Figure 1). This manuscript provides exciting evidence in the plasticity of cancer cell differentiation in response to specific treatments. It also supports the importance of combinational therapy to counteract treatment resistant pathways and the concept of targeting cancer stem cell-specific pathways that may have distinct resistance mechanisms to the bulk tumor populations.

 

 

Fig 1. Schematic Proposed Mechanism of FOXP3Δ3 in Bladder Cancer and in Treatment Resistance.

During bladder cancer development, FOXP3Δ3, which is enriched in the cancer initiating population (depicted in purple) gives rises to progressive bladder cancer. Treatment with cisplatin-based chemotherapy will destroy the bulk tumor cells (depicted in orange), but further augments FOXP3Δ3 expression in the cancer initiating population which mediates chemotherapy resistance.

 

References:

  1. Meeks, J. J. et al. A systematic review of neoadjuvant and adjuvant chemotherapy for muscle-invasive bladder cancer. Eur. Urol. 62, 523–33 (2012).
  2. Choi, W. et al. Identification of Distinct Basal and Luminal Subtypes of Muscle-Invasive Bladder Cancer with Different Sensitivities to Frontline Chemotherapy. Cancer Cell 25, 152–165 (2014).
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  4. Zuo, T. et al. FOXP3 is an X-linked breast cancer suppressor gene and an important repressor of the HER-2/ErbB2 oncogene. Cell 129, 1275–1286 (2007).
  5. Wang, L. et al. Somatic Single Hits Inactivate the X-Linked Tumor Suppressor FOXP3 in the Prostate. Cancer Cell 16, 336–346 (2009).